TRAF4 acts as a fate checkpoint to regulate the adipogenic differentiation of MSCs by activating PKM2
TRAF4 acts as a fate checkpoint to regulate the adipogenic differentiation of MSCs by activating PKM2
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TRAF4作为命运检查点通过激活PKM2调节MSC的成脂分化
DOI：
10.1016/j.ebiom.2020.102722
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发表时间：
2020-04-01
期刊：
影响因子：
11.1
通讯作者：
Shen, Huiyong
中科院分区：
文献类型：
--
作者：
Cen, Shuizhong;Li, Jinteng;Shen, Huiyong
背景：间充质干细胞(MSCs)可选择性分化为脂肪细胞或成骨细胞，多种分子控制着MSCs的命运。了解这些关键检查点对诱导临床应用中特定的MSC分化能力有很大帮助。本研究旨在探讨肿瘤坏死因子受体相关因子4(TRAF4)对MSC成脂分化的影响。方法：采用Western blotting和实时定量聚合酶链式反应检测TRAF4在成脂分化过程中的表达。构建慢病毒来调节TRAF4的表达，油红O染色和免疫印迹法检测TRAF4在脂肪形成中的作用，通过将MSC-Matrigel混合物植入裸鼠体内证实了这一点。用Western blotting检测激活的信号通路，用特异性的抑制剂和激动剂清除关键信号通路的作用。此外，通过免疫共沉淀，发现丙酮酸激酶同工酶M2(PKM2)与TRAF4相互作用，并进一步探索其结合和功能结构域。最后，用RNA结合蛋白免疫沉淀法和Western blotting检测N6-甲基腺苷是否介导了TRAF4在成脂分化过程中表达的降低。结果表明，TRAF4在体外和体内都对MSC的脂肪生成具有负性调节作用。从机制上讲，我们揭示了TRAF4与PKM2结合，激活PKM2的激酶活性，进而激活β-连环蛋白信号转导，进而抑制脂肪生成。此外，TRAF4在成脂过程中的下调受ALKBH5介导的N6-甲基腺苷RNA去甲基化的调节。解释：TRAF4通过激活PKM2激酶活性负性调节MSCs的成脂过程，这可能是微调脂肪-成骨分化平衡的检查点，提示TRAF4可能是MSCs临床应用的新靶点，也可能揭示骨代谢疾病的潜在机制。(C)2020年提交人(S)。爱思唯尔出版公司(Elsevier B.V.)
Background: Mesenchymal stem cells (MSCs) selectively differentiate into adipocytes or osteoblasts, and several molecules control the fate determination of MSCs. Understanding these key checkpoints greatly contributes to the ability to induce specific MSC differentiation for clinical applications. In this study, we aimed to explore whether TNF receptor-associated factor 4 (TRAF4) affects MSC adipogenic differentiation, which we previously reported that could positively regulated the osteogenic differentiation.Methods: Western blotting and Real-time Polymerase Chain Reaction were used to detected the expression pattern of TRAF4 during adipogenic differentiation. Lentivirus was constructed to regulate TRAF4 expression, and oil red O staining and Western blotting were used to assess its role in adipogenesis, which was confirmed in vivo by implanting an MSC-matrigel mixture into nude mice. Western blotting was used to detect the activated signaling pathways, and a specific inhibitor and agonist were used to clear the roles of the key signaling pathways. Additionaly, Co-Immunoprecipitation was conducted to find that Pyruvate kinase isozyme type M2 (PKM2) interacts with TRAF4, and to further explore their binding and functional domains. Finally, an RNA-binding protein immunoprecipitation assay and Western blotting were used to detect whether N6-methyladenosine mediates the decreased TRAF4 expression during adipogenic differentiation.Findings: The results demonstrated that TRAF4 negatively regulates MSC adipogenesis in vitro and in vivo. Mechanistically, we revealed that TRAF4 binds to PKM2 to activate the kinase activity of PKM2, which subsequently activates beta-catenin signaling and then inhibits adipogenesis. Furthermore, TRAF4 downregulation during adipogenesis is regulated by ALKBH5-mediated N6-methyladenosine RNA demethylation.Interpretation: TRAF4 negatively regulates the adipogenesis of MSCs by activating PKM2 kinase activity, which may act as a checkpoint to fine-tune the balance of adipo-osteogenic differentiation, and suggests that TRAF4 may be a novel target of MSCs in clinical use and may also illuminate the underlying mechanisms of bone metabolic diseases. (C) 2020 The Author( s). Published by Elsevier B.V.